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Am. J. Hum. Genet. 64:218–224, 1999
Rapid Clearance of Fetal DNA from Maternal Plasma
Y. M. Dennis Lo,1 Jun Zhang,1 Tse N. Leung,2 Tze K. Lau,2 Allan M. Z. Chang,2 and
N. Magnus Hjelm1
Departments of 1Chemical Pathology and 2Obstetrics and Gynecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin,
New Territories, Hong Kong
Summary
Fetal DNA has been detected in maternal plasma during
pregnancy. We investigated the clearance of circulating
fetal DNA after delivery, using quantitative PCR analysis
of the sex-determining region Y gene as a marker for
male fetuses. We analyzed plasma samples from 12
women 1–42 d after delivery of male babies and found
that circulating fetal DNA was undetectable by day 1
after delivery. To obtain a higher time-resolution picture
of fetal DNA clearance, we performed serial sampling
of eight women, which indicated that most women
(seven) had undetectable levels of circulating fetal DNA
by 2 h postpartum. The mean half-life for circulating
fetal DNA was 16.3 min (range 4–30 min). Plasma nucleases were found to account for only part of the clearance of plasma fetal DNA. The rapid turnover of circulating DNA suggests that plasma DNA analysis may
be less susceptible to false-positive results, which result
from carryover from previous pregnancies, than is the
detection of fetal cells in maternal blood; also, rapid
turnover may be useful for the monitoring of feto-maternal events with rapid dynamics. These results also
may have implications for the study of other types of
nonhost DNA in plasma, such as circulating tumor-derived and graft-derived DNA in oncology and transplant
patients, respectively.
Introduction
The two-way transfer of nucleated cells between the
mother and fetus is now a well-established phenomenon
(Walknowska et al. 1969; Lo et al. 1989, 1996). In clinical use, the transfer of fetal cells into maternal blood
Received August 12, 1998; accepted for publication October 23,
1998; electronically published December 11, 1998.
Address for correspondence and reprints: Dr. Y. M. Dennis Lo, Department of Chemical Pathology, Chinese University of Hong Kong,
Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.
E-mail: [email protected]
䉷 1999 by The American Society of Human Genetics. All rights reserved.
0002-9297/99/6401-0028$02.00
218
provides a source of fetal genetic material for noninvasive prenatal diagnosis (Bianchi et al. 1990; Simpson
and Elias 1995; Cheung et al. 1996). After delivery, most
fetal cells are cleared by 2–3 mo postpartum (Lo et al.
1993; Hamada et al. 1994; Thomas et al. 1995). By the
use of cell sorting and sensitive PCR assays, fetal hematopoietic progenitor cells have been shown to persist
in some women, even decades after delivery (Bianchi et
al. 1996). The latter phenomenon has been proposed to
be associated with certain autoimmune disorders (Artlett
et al. 1998; Nelson et al. 1998).
We have shown recently that, in addition to the presence of fetal cells in maternal blood, cell-free fetal DNA
is also present in maternal circulation (Lo et al. 1997).
By means of a quantitative PCR assay, fetal DNA has
been demonstrated to be present in high concentrations
in maternal plasma (Lo et al. 1998a). This observation
suggests that plasma fetal DNA analysis may have clinical applications in the noninvasive prenatal diagnosis
of certain disorders, including sex-linked diseases and
fetal hemolytic disease resulting from Rh blood group
incompatibility (Lo et al. 1997; Bianchi 1998). Little is
known about the parameters governing the level of circulating fetal DNA, except that it tends to increase as
gestation progresses, especially toward the end of pregnancy (Lo et al. 1998a).
In the present study, we investigated the clearance of
fetal DNA from maternal plasma after delivery. If a
steady-state situation is assumed, the kinetics of fetal
DNA clearance will allow estimation of the rate of fetal
DNA release into maternal circulation. The rate of clearance will also provide information as to the applicability
of fetal DNA measurement in the study of the dynamic
processes involved in the handling of circulating DNA
during pregnancy. We also aim to obtain data about the
role of plasma nucleases in the clearance of fetal DNA
from maternal circulation.
Our study relied on the recent development of a realtime quantitative PCR assay for fetal-derived DNA sequences in maternal plasma (Lo et al. 1998a). For fetal
DNA detection, we used the sex-determining region Y
(SRY) gene on the Y chromosome, as a marker for male
fetuses. In the first stage of our project, we studied 12
women with male babies, at various times after delivery.
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Lo et al.: Rapid Clearance of Circulating Fetal DNA
Having established that fetal DNA is cleared very rapidly
after parturition, we then carried out serial sampling of
8 women, to obtain a high time-resolution picture of
fetal DNA clearance. These data are valuable for furthering our understanding of the feto-maternal transfer
of nucleic acids.
mediate processing. Blood samples were centrifuged at
3,000 g, and plasma was removed and transferred into
plain polypropylene tubes. The plasma samples were recentrifuged at 3,000 g, and the supernatants were collected into fresh polypropylene tubes. The samples were
stored at ⫺20⬚C until further processing.
Subjects and Methods
DNA Extraction from Plasma Samples
Subjects
Pregnant women attending the Department of Obstetrics and Gynecology at the Prince of Wales Hospital,
Shatin, Hong Kong, were recruited, and informed consent was obtained. Approval for the study was obtained
from the Research Ethics Committee of the Chinese University of Hong Kong. For the first part of the project,
healthy pregnant women were recruited just after the
onset of labor. Samples of maternal peripheral blood
(5–10 ml) were collected into tubes containing EDTA.
Only subjects who subsequently delivered male babies
vaginally were followed-up, and blood was obtained by
venesection, at a single time point, after delivery. Ten
subjects who subsequently delivered female fetuses were
recruited as negative controls. Blood samples were obtained from subjects at one of the following time points:
6 wk, 1 wk, or 1 d after delivery. For the second part
of the project, subjects were recruited just before undergoing elective cesarean section. These women were
free of any medical disease or any antenatal complications, and their elective cesarean sections were indicated
because of previous cesareans or (in one case) because
of abnormal fetal lie. The single case of abnormal fetal
lie (subject S3) had not been subjected to external cephalic version. An ultrasound scan was done, to ascertain fetal sex (subsequently confirmed at delivery), and
only subjects determined to be carrying male babies were
studied. Maternal peripheral blood samples (5 ml) were
collected, before cesarean section, into an EDTA-containing tube. After delivery of the baby, 2 ml maternal
peripheral blood was collected into an EDTA tube at 5,
15, 30, 45, 60, and 120 min postpartum. A total of 8
subjects with male babies were studied. For the 10 subjects recruited for the study of the role of plasma nucleases, 5 ml maternal blood was collected into a plain
tube before cesarean section and at 2 h after delivery.
Sample Preparation
Blood samples from subjects, obtained at a single postdelivery time point, were processed as described elsewhere (Lo et al. 1998a). For subjects recruited for the
serial sampling project and for the study of the role of
plasma nucleases, a researcher was present at the time
of collection of the pre- and postdelivery samples, which
were transported instantly to the laboratory, for im-
DNA from plasma samples was extracted by use of a
QIAamp Blood Kit (Qiagen); the blood and body fluid
protocol, as recommended by the manufacturer, was followed (Chen et al. 1996). For subjects whose blood was
sampled at a single postdelivery time point, an 800-ml
plasma sample was used for DNA extraction; 200 ml
plasma was used, for DNA extraction, for subjects
whose blood was sampled at multiple postdelivery time
points and for subjects recruited for the study of the role
of plasma nucleases.
Real-Time Quantitative PCR
Real-time quantitative PCR analysis was done as described elsewhere, by use of a 7700 Sequence Detector
(PE Applied Biosystems), which is essentially a combined
thermal cycler and fluorescence detector with the ability
to monitor the progress of individual PCR reactions optically (Heid et al. 1996; Lo et al. 1998a). The amplification and product reporting system used is based on
the 5 nuclease assay (TaqMan assay; Perkin-Elmer; Holland et al. 1991). In this system, apart from the two
amplification primers as used in conventional PCR, a
dual-labeled fluorogenic hybridization probe is also included (Lee et al. 1993; Livak et al. 1995). One fluorescent dye serves as a reporter (6-carboxyfluorescein),
and its emission spectrum is quenched by a second fluorescent dye (6-carboxy-tetramethylrhodamine). During
the extension phase of PCR, the 5-to-3 exonuclease activity of the Taq DNA polymerase cleaves the reporter
from the probe, thus releasing it from the quencher, resulting in an increase in fluorescent emission at 518 nm.
This sequence detector is able to measure the fluorescent
spectra of the 96 amplification wells continuously during
DNA amplification, and data are captured onto a Macintosh computer (Apple Computer).
Primer and probe sequences for the SRY and the bglobin genes were as described elsewhere (Lo et al.
1998a). TaqMan amplification reactions were set up in
a reaction volume of 50 ml, with components (except
TaqMan probes and amplification primers) supplied in
a TaqMan PCR Core Reagent Kit (Perkin-Elmer).
TaqMan probes were custom-synthesized by PE Applied
Biosystems. PCR primers were synthesized by Life Technologies. Each reaction contained 5 ml 10 # buffer A;
300 nM each amplification primer; 100 nM of the corresponding TaqMan probe; 4 mM MgCl2; 200 mM each
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Am. J. Hum. Genet. 64:218–224, 1999
dATP, dCTP, and dGTP; 400 mM dUTP; 1.25 U
AmpliTaq Gold; and 0.5 U AmpErase uracil N-glycosylase. Five microliters of the extracted plasma DNA were
used for amplification. DNA amplifications were performed in 96-well reaction plates that were frosted by
the manufacturer to prevent light reflection and that
were closed with caps designed to prevent light scattering
(Perkin-Elmer). Each sample was analyzed in duplicate.
A calibration curve was run in parallel and in duplicate
with each analysis. The conversion factor of 6.6 pg
DNA/cell (Saiki et al. 1988) was used to express the
results as copy numbers.
An identical thermal profile was used for both the SRY
and b-globin TaqMan systems. Thermal cycling was initiated with a 2-min incubation at 50⬚C for the uracil
N-glycosylase to act, followed by a first denaturation
step of 10 min at 95⬚C. Next, 40 cycles of 95⬚C for 15
s and 60⬚C for 1 min were performed.
Amplification data collected by the 7700 Sequence
Detector and stored in the Macintosh computer were
then analyzed by means of the Sequence Detection System software (PE Applied Biosystems). The mean quantity of each duplicate was used for further concentration
calculation. The concentration, expressed in copies per
milliliter, was calculated by use of the equation
C⫽Q
(VV )(V1 ) ,
DNA
PCR
ext
where C ⫽ target concentration in plasma (copies per
milliliter); Q ⫽ target quantity (copies), determined by
sequence detector in a PCR; VDNA ⫽ total volume of
DNA, obtained after extraction, typically 50 ml per Qiagen extraction; VPCR ⫽ volume of DNA solution used
for PCR, typically 5 ml; and Vext ⫽ volume of plasma
extracted, either 0.2 ml or 0.8 ml.
Results
Subjects Studied before Delivery and at One Time
Point after Delivery
Twelve subjects with male babies were studied. Plasma
samples collected before delivery revealed the presence
of fetal-derived SRY sequences, in all instances (table 1).
Fetal DNA was not detected in any of the postpartum
samples with collection times of 1–42 d after delivery.
b-globin TaqMan PCR was done for all pre- and postdelivery samples, which demonstrated the presence of
amplifiable DNA in all instances. None of the predelivery plasma samples taken from the 10 women who gave
birth to female fetuses had detectable SRY signals.
Sequential Follow-Up after Delivery
Eight women were recruited just before undergoing
elective cesarean section. Plasma samples were obtained
before cesarean section and at 5, 15, 30, 45, 60, and
120 min after delivery. b-globin TaqMan PCR was done
for all pre- and postdelivery samples, which demonstrated the presence of amplifiable DNA in all instances.
The fetal DNA concentration in maternal plasma was
determined by use of real-time quantitative PCR (fig. 1).
In three instances (subjects S1, S3, and S6), there was a
rise in plasma fetal DNA concentration shortly after delivery, at 5 min, compared with the predelivery value.
Seven of the eight women had no detectable plasma fetal
DNA by 120 min postpartum. The remaining subject
(S6) had 90% of maternal plasma fetal DNA cleared by
120 min after delivery. The mean time taken to reduce
the peak plasma fetal DNA concentration by 50% was
16.3 min (range 4–30 min).
Role of Plasma Nucleases
To study the role of plasma nucleases in the clearance
of fetal DNA from maternal plasma, we collected pe-
Anticontamination Measures
Strict precautions against PCR contamination were
taken (Kwok and Higuchi 1989). Aerosol-resistant pipette tips were used for all liquid handling. Separate
areas were used for setup of amplification reactions, addition of DNA template, and execution of amplification
reactions. The 7700 Sequence Detector offered an extra
level of protection, because its optical detection system
obviated the need to reopen the reaction tubes after completion of the amplification reactions, thus minimizing
the possibility of carryover contamination. In addition,
the TaqMan assay included a further anticontamination
measure in the form of preamplification treatment with
uracil N-glycosylase, which destroyed uracil-containing
PCR products (Longo et al. 1990). Multiple negativewater blanks were included in each analysis.
Table 1
Detection of Fetal DNA in Maternal Plasma
Case
Predelivery
(copies/ml)
Postdelivery
(copies/ml)
Time postdelivery
(days)
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
29.0
112.5
80.0
337.5
27.5
41.3
33.8
50.3
63.5
176.1
99.4
3,024.0
0
0
0
0
0
0
0
0
0
0
0
0
42
42
7
7
7
7
7
1
1
1
1
1
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Lo et al.: Rapid Clearance of Circulating Fetal DNA
Figure 1
Sequential follow-up of circulating fetal-derived SRY sequence in plasma of women, after cesarean section. Time (min) after
delivery is plotted on the X-axis, and the plasma SRY level (copies/ml) is plotted on the Y-axis. Time of “0” denotes the predelivery sample.
ripheral blood samples, before cesarean section, from 10
women carrying male fetuses. The blood samples were
collected into plain tubes, transported instantly to the
laboratory, and processed immediately. After incubation
at 37⬚C for 2 h, samples from three subjects (N1, N5,
and N9) had plasma fetal DNA concentrations 190%
of preincubation values (fig. 2). The remaining seven
subjects’ samples had concentrations with a range of
31%–74% of the values before incubation (fig. 2). At 2
h after delivery, a second peripheral blood sample was
obtained from each of these women. Nine subjects did
not have detectable circulating fetal DNA in their
plasma. The remaining subject (N6) had a circulating
fetal DNA level at 12% of that of the predelivery sample.
Discussion
In this study, we present the first data on the clearance
of fetal DNA from maternal plasma. Previous work on
the clearance of fetal cells from maternal blood indicates
that the clearance in most subjects takes place over a
period of weeks (Lo et al. 1993; Hamada et al. 1994;
Thomas et al. 1995) and that, in certain persons, fetal
hematopoietic progenitors are detectable, even decades
after delivery (Bianchi et al. 1996). These data prompted
us to start our investigation into fetal DNA clearance at
6 wk (42 d) after delivery. When no fetal DNA was
detectable in maternal plasma at 6 wk, we gradually
reduced the time interval studied, to gauge the time
222
Figure 2
Effect of plasma nucleases on the clearance of circulating fetal DNA. Blood samples were obtained from 10 women
(N1–N10), before delivery. Plasma SRY levels (copies/ml) were assayed, both before and after incubation, at 37⬚C for 2 h.
frame of fetal DNA clearance. This approach allowed
us to determine that fetal DNA is cleared very rapidly
from maternal plasma, with the first phase of our project
indicating that fetal DNA became undetectable by 1 d
after delivery.
In the second part of the project, we used serial blood
sampling of 8 pregnant women carrying male fetuses
who were delivered by cesarean section. We chose
women undergoing cesarean section, because the time
of delivery could be easily and accurately determined in
these subjects, and because the time of blood sampling
could be planned in advance. These data confirm the
rapid clearance of fetal DNA from maternal plasma.
Indeed, seven of the eight women had undetectable
plasma fetal DNA by 2 h postpartum. The single subject
(S6) with detectable plasma fetal DNA had 90% of circulating fetal DNA cleared by 2 h postpartum. In three
instances (subjects S1, S3, and S6), there was a noticeable
rise in plasma fetal DNA concentration shortly after delivery, at 5 min, compared with the predelivery value.
This phenomenon may be a result of delivery-associated
processes operating in these subjects, which could have
resulted in increased direct liberation of fetal DNA or
in increased indirect fetal DNA release, secondary to the
destruction of fetal nucleated cells, after their entry into
maternal blood (e.g., because of feto-maternal hemorrhage). Review of the obstetric notes indicates that, of
the eight women, three (subjects S1, S3, and S8) underwent manual removal of the placenta, whereas the re-
Am. J. Hum. Genet. 64:218–224, 1999
maining five had the placenta removed by the more gentle procedure of controlled cord traction. It is possible
that the potentially more traumatic procedure of manual
removal precipitated a release of fetal DNA into the
maternal circulation in the two examples in which the
largest rise in fetal DNA after the procedure was demonstrated (subjects S1 and S3). Study of the curves revealed that fetal DNA clearance appeared to occur in
two phases, with different kinetics: an initial, more rapid
phase, followed by a slower phase. This observation suggests that more than one mechanism may be involved
in the clearance of circulating fetal DNA.
Our data demonstrate the rapid clearance of circulating fetal DNA after delivery. Assuming there are no
abrupt changes in circulating fetal DNA clearance associated with delivery, one may be able to extrapolate
our findings to the predelivery state, so as to gain mechanistic insights about the physiology of DNA transfer
from the fetus to the mother. On the basis of this assumption, in spite of the rapid clearance of fetal DNA
in maternal plasma, previous work has shown that fetal
DNA is present in high concentrations and is readily
detectable in maternal circulation during pregnancy (Lo
et al. 1998a). These observations suggest that, to maintain a steady state, fetal DNA must be liberated in large
quantities into maternal circulation. Our previous work
has shown that the mean maternal plasma fetal DNA
concentration in the third trimester of pregnancy is 292
copies/ml (Lo et al. 1998a). If a plasma volume of 2,500
ml is assumed, the total amount of fetal DNA that is
present in maternal circulation is 292 # 2,500 copies ⫽
7.3 # 10 5 copies. With a mean plasma half-life of 16.3
min, 50% of these 7.3 # 10 5 copies (i.e., 3.65 #
10 5 copies) will be cleared in 16.3 min. The mean fetal
DNA clearance rate is estimated to be 2.24 # 10 4 copies/min. In the steady state, the liberation rate of fetal
DNA should be equal to this clearance rate. Therefore,
our calculations suggest that fetal DNA is liberated at
a mean rate of 2.24 # 10 4 copies/min into the maternal circulation. It should be noted that these calculations
represent an average only and that there are significant
variations in the fetal DNA clearance rate (and the liberation rate) among individual cases, as evidenced by
the range of half-lives observed. Nonetheless, these baseline data would be very useful to guide future studies
on the pathologic or physiologic parameters affecting
the liberation or clearance of fetal DNA from maternal
blood. An interesting example is pregnancies complicated by Rh blood group incompatibility, in which increased liberation of fetal DNA may occur because of
hemolysis of fetal nucleated red cells.
The rapid turnover of plasma fetal DNA implies that
its level provides an almost real-time picture of fetal
DNA production and clearance and, thus, may be useful
for monitoring feto-maternal events having rapid dy-
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Lo et al.: Rapid Clearance of Circulating Fetal DNA
namics. Fetal cells in maternal blood, the concentrations
of which change relatively slowly over the course of
weeks (Hamada et al. 1994), may not be as good a
marker for these events. One possible example is the
monitoring of the occurrence and resolution of feto-maternal hemorrhage, in conjunction with established
methods of detecting fetal hemoglobin–containing cells
(e.g., the Kleihauer test). The SRY PCR system, described
in the present study, is applicable only to pregnancies
involving male fetuses. The application of plasma PCR
to pregnancies involving female fetuses will require the
development of quantitative PCR systems that are applicable to autosomal polymorphic loci. Potential loci
suitable for this type of analysis have been described
elsewhere (Lo et al. 1996).
For prenatal diagnostic purposes, the rapid clearance
of fetal DNA in maternal plasma makes the approach
described in the present study less susceptible to falsepositive results that are caused by the persistence of fetal
DNA from one pregnancy into the next. The persistence
of fetal cells has been raised as a potential source of false
positivity when fetal cells in maternal blood are used for
noninvasive prenatal diagnosis (Hsieh et al. 1993; Bianchi et al. 1996).
Other work has demonstrated the existence of nucleases in the plasma of humans (Herriott et al. 1961).
Experiments involving the injection of purified DNA
into animals has indicated a role for plasma nucleases
in the degradation of the injected DNA (Paoletti et al.
1963; Gosse et al. 1965; Emlen and Mannik 1984). The
significance of these data with regard to the clearance
of circulating fetal DNA is unclear, because the starting
material in these animal experiments are DNA preparations that have been purified highly and that are derived from other species. To elucidate the role of plasma
nucleases in the clearance of circulating fetal DNA, we
incubated plasma from 10 pregnant women at 37⬚C,
after blood collection. We used nonanticoagulated blood
for this experiment, to avoid the potential inhibitory
effect of anticoagulants on plasma nucleases (Herriott
et al. 1961). To avoid the release of nuclease inhibitors
after blood clotting (Frost and Lachmann 1968), venesection was done in the presence of a researcher, who
immediately transported the blood samples to the laboratory, where it was processed promptly. In vitro incubation of fresh plasma samples for 2 h at 37⬚C resulted
in the incomplete removal of plasma fetal DNA in all
instances. In contrast, the analysis of a second maternal
blood sample, collected 2 h after delivery, revealed the
complete clearance of circulating fetal DNA in 9 of the
10 subjects. These data show that plasma nucleases play
only a partial role in the removal of circulating fetal
DNA in most subjects and suggest that other organ systems also contribute toward the clearance of circulating
fetal DNA. Previous work done on the basis of the in-
jection of extracted, foreign DNA into experimental animals variously has implicated the liver, spleen (Chused
et al. 1972; Emlen and Mannik 1978), and kidney
(Tsumita and Iwanaga 1963) as being involved in the
removal of circulating DNA. Further work to elucidate
the important organ systems in fetal DNA clearance may
require the study of subjects with known disorders in
the respective organ systems, such as persons with
chronic liver disease or renal failure.
The presence of fetal DNA in maternal plasma is only
one of a number of clinical scenarios in which nonhost
DNA can be found in the plasma of human subjects.
The other two situations are in oncology and transplant
patients, in whom tumor-derived and donor-derived
DNA has been demonstrated to circulate in the peripheral blood (Chen et al. 1996; Nawroz et al. 1996; Lo
et al. 1998b). It is likely that, analogous to the rapid
clearance of fetal DNA from maternal blood, tumorderived and organ donor–derived DNA may also be rapidly removed. The rapid kinetics of circulating DNA
potentially will make plasma-based molecular diagnostics very useful for monitoring dynamic changes in patients with cancer and in transplant recipients under specific clinical circumstances. One possible application is
in the monitoring of potential complications of cancer
chemotherapy, for example, the tumor lysis syndrome.
Acknowledgments
Y.M.D.L., A.M.Z.C., and N.M.H. are supported by the
Hong Kong Research Grants Council.
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